Biological processes, in particular microbial conversions, are increasingly being employed to perform a variety of useful roles. Examples of applications in which microbial conversions have been successfully utilised include microbial synthesis, the treatment of industrial effluent, the removal of contaminants from contaminated soils and groundwaters, and the treatment of contaminated gas streams for example biotreatment of air-stripped streams such as petroleum additives and the like.
Over the past decade, environmental concerns and regulatory trends have increased the need for means to remove contaminants from contaminated soils and groundwaters. Such soils and groundwaters may become contaminated through natural sources for example the leeching of salts and/or minerals from surrounding rocks into the soil or groundwater, but more commonly they become contaminated through the activities of man, with contaminants such as metals, particularly heavy metals (e.g. mercury, chromium, lead); organic compounds (e.g. solvents, petroleum, petroleum related products, pesticides), inorganic compounds (e.g. nitrates); micro-organisms (e.g. pathogenic bacteria and viruses) and radio active compounds (e.g. uranium), having entered soils and groundwaters through disposal or spillage. One such class of contaminants are branched alkyl ethers such as methyl tert-butyl ether (MTBE) which have been widely used as additives in gasoline blends and which have been found to accumulate in aquifer groundwaters so contaminating supplies of drinking water.
Accumulation of contaminants in soils and groundwaters may occur when natural attenuation of said contaminants is limited by a lack of suitable microbes, oxygen, or nutrients (e.g. inorganic phosphate and nitrogen sources). One way to treat contaminated soils and groundwaters is to contact the soils or groundwaters with a microbial culture capable of converting the contaminant into non-harmful products in a bioreactor. Bioreactors have commonly been used in conjunction with ex-situ pump-and-treat methods of bioremediation, wherein contaminated groundwater is pumped from an aquifer, treated with a microbial culture in a bioreactor, and reinjected into the aquifer or discharged above-ground. An alternative method of bioremediation is to employ an in-situ method, wherein an indigenous or non-indigenous microbial culture capable of metabolising a contaminant is injected into soils or groundwaters contaminated with said contaminant in an environment conducive to the growth and development of the culture. However, in-situ methods of bioremediation have proven difficult to control since the conditions in an aquifer are subject to change (e.g. pH, oxygen availability, nutrient availability) making it difficult to create an environment conducive to the growth and development of the microbial culture. Further drawbacks of in-situ methods of bioremediation are the potential for escape of non-indigenous microbial cultures from treatment areas, and the blocking of the aquifer with solids.
To date, the practicality of using microbial cultures to remediate contaminated soils and groundwaters has been limited in that many of the most persistent contaminants are metabolised very slowly as primary substrates and are only substantially metabolised at a satisfactory rate in co-metabolic microbial conversions. Microbial conversion of a particular substrate by a microbial culture will usually involve both catabolic and anabolic activities, wherein the microbial culture produces multi catabolic enzymatic activities that degrade the compound to intermediates which can either be used for biosynthetic purposes (anabolism) or can be mineralized to carbon dioxide as part of the energy-generating processes of a cell. In such conventional microbial conversions, it is usually the degradation of the so-called primary growth substrate that provides energy and a carbon source for microbial growth. In contrast, a co-metabolic microbial conversion will involve the catalytic activity of a few, often only a single, enzyme and the intermediates produced are not sufficiently available or suitable for the key anabolic processes required to support microbial growth. Because of this separation between catabolic activity and anabolic growth, a primary growth substrate must be supplied to support growth of microbial cultures for co-metabolic conversions, the primary growth substrate being a carbon source which the culture can use to support its growth. To date, co-metabolic microbial conversions have been considered unsuitable for use in in-situ methods of bioremediation as it is often impractical and/or undesirable to add a primary growth substrate into aquifer groundwater.
A further limitation on the use of co-metabolic microbial conversions is that the primary growth substrate can be toxic to the microbial culture when provided at too high a concentration. For example, when the primary growth substrate is a hydrocarbon and the microbial culture exists in an aqueous medium, the addition of the hydrocarbon growth substrate at a concentration greater than the solubility of the hydrocarbon in water can kill culture cells. This is especially problematic as a high supply rate and/or concentration of growth substrate is often required in order to maintain a biomass of microbial culture sufficient to metabolise the quantity of substrate, e.g. contaminant, to be treated, and that the bioreactors presently available are inadequate for the maintenance of a microbial culture in such circumstances.
U.S. Pat. No. 5,227,136 discloses a bioreactor vessel comprising a tank adapted to receive and contain a slurry, a mechanical mixing means fitted in the tank, an air supply means which involves the introduction of minute air bubbles near the bottom region of the tank by a plurality of elastic membrane diffusers (col. 3, line 20 to 32) and a means of re-circulating exhaust gas stream back into the reactor contained slurry by means of the diffusers (col. 4, line 6 to 11). In use, slurry containing minerals, soils and/or sludges which have been contaminated by toxic organic substances are delivered to the tank where they are directly contacted with and degraded by a biomass. Maintaining a high biomass concentration in the reactor is said to be a task requiring the use of equipment ancillary to the bioreactor (col. 4, lines 1 to 5) and in a preferred embodiment of the invention a biomass-carrying medium is added to the slurry contained in the tank to assist in maintaining a maximum biomass concentration (col. 10 lines 10 to 16). Whilst it is mentioned at column 9, lines 54 to 57 that another gas which may act as a co-metabolite in a biodegradation process may be added with the re-circulating gas stream, from the teaching of U.S. Pat. No. 5,227,136 the skilled person would conclude that in operation it is necessary to provide further microbial culture to the bioreactor vessel together with each batch of slurry to be remediated.
WO 96/34087 discloses a bioreactor comprising a component defining chamber in which are disposed fluid treatment cells, a liquid-permeable membrane which separates the chamber from a first channel in which fluid to be treated flows, and a gas-permeable membrane separating the chamber from a second channel in which an oxygenous gas flows. The chamber preferably comprises two cell layers separated by a permeable layer. The bioreactor is for the study of cell cultures, in particular tissue cell cultures, and is particularly developed for use in specialist medical applications such as an artificial liver or a device for facilitating the functioning of a failing liver. Whilst the bioreactor of WO 96/34087 comprises two separate membranes through which oxygen and fluid to be treated may be simultaneously supplied to the cells, there is no mention of a separate means to supply the treatment cells with a primary growth substrate (i.e. a source of carbon and energy) other than the fluid to be treated. Therefore, from the teaching of WO 96/34087 a skilled person would be led to conclude that the bioreactor disclosed therein is suitable only for conventional or non co-metabolic microbial conversions, in particular conversions using tissue cultures.
It is apparent therefore that there is a need for a bioreactor which may be used with both conventional microbial conversions and co-metabolic microbial conversions and which may be adapted for use in in-situ methods of bioremediation. cl SUMMARY OF THE INVENTION
The present invention provides a bioreactor for microbial conversion of at least one conversion substrate which comprises a treatment zone to accommodate when in use a solution of said at least one conversion substrate, a culture holding zone to accommodate when in use a microbial culture capable of metabolising said at least one conversion substrate, a source of primary growth substrate for the microbial culture, a first permeable membrane forming an interface between the treatment zone and the culture holding zone, and a second permeable membrane forming an interface between the source of primary growth substrate and the culture holding zone, the first permeable membrane being of a material which will allow passage of the at least one conversion substrate from the treatment zone to the culture holding zone whilst being impermeable to the microbial culture, the second permeable membrane being of a material permeable to the primary growth substrate but substantially impermeable to water.